The complete chloroplast genome of Oxytropis ochrocephala Bunge 1874 (Fabaceae) and its phylogenetic analysis

Abstract This study presents the first-ever complete chloroplast (cp) genome sequence of Oxytropis ochrocephala Bunge 1874, a member of the Fabaceae family. The cp genome spans 126,996 base pairs and includes 109 genes, comprising 76 protein-coding genes, 29 tRNA genes, and four rRNA genes. Notably, the genome lacks an inverted repeat (IR) region. Additionally, we constructed phylogenetic trees for 34 species within Trib. Galegeae, employing both maximum likelihood (ML) and Bayesian inference (BI) methods. These analyses robustly support the monophyly of the Oxytropis species, evidenced by high bootstrap values (BP = 100) and posterior probabilities (PP = 1). Within this clade, O. ochrocephala exhibits a sister relationship with other Oxytropis species. Our findings provide valuable insights into the genetic makeup and evolutionary relationships of O. ochrocephala within the Galegeae tribe.


Introduction
Oxytropis ochrocephala Bunge 1874, a perennial medicinal herb (Zhou et al. 2020;Xue et al. 2022;Yu 2022) within the Oxytropis genus of the Galegeae tribe in the Fabaceae family, exhibits notable ecological and nutritional significance.Characterized by a robust root system and strong reproductive capacity, this species demonstrates remarkable resilience to environmental stressors such as cold, drought, salinity, alkalinity, and sand winds.This resilience contributes positively to the restoration of fragile ecosystems.Nutritional analyses reveal that O. ochrocephala's crude fiber and protein content are comparable to that of Medicago sativa, suggesting its potential as a high-quality forage resource for livestock following appropriate detoxification (Shen and Mo 2013).
Previous research on O. ochrocephala has primarily focused on aspects like seed germination (Chen et al. 2017), chemical composition (Zhou et al. 2020), pharmacological activity (Tan et al. 2016), genetic structure (He et al. 2015), and endophytic bacterial diversity (Jiang et al. 2019;Zhang et al. 2022).However, there is a noticeable gap in the literature regarding the structural characteristics and phylogeny of its complete chloroplast (cp) genome.The scarcity of cp genome resources among the numerous Oxytropis species further limits related studies.
Addressing this gap, our study involves sequencing and analyzing the cp genome of O. ochrocephala using advanced high-throughput sequencing technology and bioinformatics methods.Our research elucidates the cp genome structure and clarifies the phylogenetic relationships within the Oxytropis genus.This study aims to provide a scientific foundation for species identification and understanding the phylogenetic relationships within Oxytropis.

Materials
For our study, we selectively collected natural, healthy, and pest-free leaves of O. ochrocephala from Delingha City, located in the Haixi Mongolian and Tibetan Autonomous Prefecture of Qinghai Province, China.The specific coordinates of our collection site were 37.37 � N, 97.37 � E, at an altitude of 2952 m (Figure 1).To ensure the preservation of the samples' integrity, the leaves were immediately placed into silica gel for desiccation.The specimen was deposited at Qinghai Provincial Key Laboratory of Physical Geography and Environmental Processes of Qinghai Normal University, Xining, China under the voucher number QTP-LJQ-CHNQ-024-4013 (contact: Xu Su, xusu8527972@126.com).

Methods
Total genomic DNA was extracted from the collected samples using a modified CTAB method (Doyle and Doyle 1987).To assess DNA integrity, concentration, and purity, we employed 1.0% agarose gel electrophoresis and a Nanodrop 2000 spectrophotometer, respectively.Following quality assurance, DNA meeting our criteria was utilized for library construction via PCR amplification.The library was then sequenced using next-generation sequencing (NGS) on the Illumina NovaSeq 6000 platform at Beijing Novogene Technology Company Limited, Beijing, China.
Furthermore, 34 cp genome sequences of species from Trib.Galegeae, as referenced in Figure 2 with GenBank accession numbers, were aligned using MAFFT (Katoh et al. 2002).
The aligned sequences were refined using trimAl (Salvador et al. 2009).Phylogenetic trees involving these 34 species were constructed, using Sophora alopecuroides (MK114100) as an outgroup.The maximum-likelihood (ML) trees were generated via IQ-TREE (Nguyen et al. 2015), selecting TVM þ F þ R4 as the optimal model and setting the bootstrap value to 5000.The Bayesian inference (BI) tree was built using MrBayes (Ronquist et al. 2012)
Typically, the cp genomes of angiosperms exhibit a quadripartite structure, consisting of a pair of inverted repeats (IRs), a LSC region, and a SSC region (Xu et al. 2023).In addition, the IR region plays an important role in stabilizing the structure of the cp genome, and the reduction, expansion and loss of the reverse repeat region directly affect the change of the cp genome structure (Song et al. 2017).However, the cp genome of O. ochrocephala deviates from this norm because it lacks quadripartite structure due to the absence of IR region, which may be the main reason for its shorter cp genome length, lower GC content and lower number of coding genes.Meanwhile, the characteristic absence of an IR region has also been observed in other cp genomes of Oxytropis genus (Su et al. 2019;Liu et al. 2021).This leads to the hypothesis that the loss of an IR region may be a common feature of the cp genomes in the Oxytropis genus.It may also be related to the process of species differentiation of Oxytropis.
Utilizing the cp genome sequences of 34 species from Trib.Galegeae, we constructed phylogenetic trees using both ML and BI methods, with Sophora alopecuroides serving as the outgroup for each analysis.The resultant phylogenetic trees, as illustrated in Figure 2(A), demonstrated identical topologies with high support values (100/1) across the majority of branches.Notably, the Oxytropis genus formed a monophyletic group in both trees.Within this group, O. ochrocephala displayed a sister relationship to the other species in the Oxytropis genus, as depicted in Figure 2(B).

Conclusions
This study successfully sequenced the cp genome of O. ochrocephala for the first time, revealing a genome length of 126,996 base pairs and an average GC content of 34.3%.The genome comprises 109 genes, which include 76 protein-coding genes, 29 tRNA genes, and four rRNA genes.Notably, the cp genome exhibits a unique circular structure, distinguished by the absence of an IR region.Phylogenetic analysis based on this genome sequence indicates that O. ochrocephala shares a sister relationship with other species within the Oxytropis genus.

Figure 3 .Figure 2 .
Figure 3. Gene map of the cp genome of O. ochrocephala.The species name and GC content are shown in the top left corner.The map contains six tracks in default.From the center outward, the first track shows the dispersed repeats.The dispersed repeats consist of direct and palindromic repeats, connected with red and green arcs.The second track shows the long tandem repeats as short blue bars.The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors.These colors correspond to the type and description of each repeat, with black representing complex repeats, green for repeat unit size 1 and yellow for size 2. The fourth track displays the genome length.The fifth track shows the GC content along the genome, while the sixth track sounds the genes.The gene names are followed by optional information about codon usage bias and color-coded based on their functional classification.The inner genes are transcribed clockwise, and the outer genes are transcribed anticlockwise.The functional type of the genes is shown in the bottom left corner.